Nucl. Acids Res.-2011-Sakurai-1510-25

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A role for human Dicer in pre-RISC loadingof siRNAs

Kumi Sakurai1,2, Mohammed Amarzguioui1,3, Dong-Ho Kim1,4, Jessica Alluin1,

Bret Heale

1,2,5

, Min-sun Song

1

, Anne Gatignol

6

, Mark A. Behlke

7

and John J. Rossi

1,2,

*1Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, 1450 East Duarte Road,

Duarte, CA 91010, 2Irell and Manella Graduate School of Biological Sciences, City of Hope, 1450 East Duarte

Road, Duarte, CA 91010, USA, 3The Biotechnology Centre of Oslo, Gaustadalleen 21, 0349 Oslo, Norway,4Genolution Pharmaceuticals, Inc. Songpa-gu, Seoul, 138-040, Korea,5Human Genetics Unit, MRC, Western

General Hospital, Crew Road, Edinburgh Scotland, UK, 6Virus-cell Interactions Laboratory, Lady Davis Institute for

Medical Research, McGill University, Montreal, Canada and 7Integrated DNA Technologies, Inc. 1710 Commercial

Park, Coralville, IA 52241, USA

Received February 27, 2010; Revised August 19, 2010; Accepted September 7, 2010

ABSTRACTRNA interference is a powerful mechanism for

sequence-specific inhibition of gene expression. It

is widely known that small interfering RNAs

(siRNAs) targeting the same region of a

target-messenger RNA can have widely different

efficacies. In efforts to better understand the siRNA

features that influence knockdown efficiency, we

analyzed siRNA interactions with a high-molecular

weight complex in whole cell extracts prepared

from two different cell lines. Using biochemical

tools to study the nature of the complex, our results

demonstrate that the primary siRNA-binding protein

in the whole cell extracts is Dicer. We find that Dicer

is capable of discriminating highly functional versus

poorly functional siRNAs by recognizing the

presence of 2-nt 30 overhangs and the thermodynam-

ic properties of 24 bp on both ends of effective

siRNAs. Our results suggest a role for Dicer in

pre-selection of effective siRNAs for handoff to

Ago2. This initial selection is reflective of the overall

silencing potential of an siRNA.

INTRODUCTION

RNA interference (RNAi) is an evolutionarily conserved

process mediated by a multi-component ribonucleoproteincomplex called the RNA induced silencing complex(RISC) (12). RNAi can be described as having at leasttwo well-defined steps: the initiation step, where the ribo-nuclease (RNase) III enzyme Dicer processes dsRNAsinto 2122-nt-long duplexes (3), and the effector step, in

which Argonaute 2 (Ago 2), a core endonuclease of theRISC, executes RNAi (46). Two classes of small regula-tory RNAssmall interfering RNAs (siRNAs) andmicroRNAs (miRNAs)are shown to associate withRISC as RNAi trigger molecules. SiRNAs, 2123 ntfully complementary double-stranded RNAs (dsRNAs)with 2-nt 30 overhangs, guide sequence-specific degrad-ation of complementary messenger RNAs onceincorporated into RISC (710). In contrast, miRNAsform imperfect duplexes with the target mRNAs, mostoften in the 30-UTR, and the miRNA-containing RISCblocks translation or leads to destabilization of thetargeted message (1118).

The RNAse III family member Dicer, along with

co-factor RNA-binding proteins, processes long dsRNAsand pre-miRNAs into the functional 2123-nt siRNA ormiRNA duplexes. The functional roles of Dicer have beenmost well studied in Drosophila where two distinctenzymes are involved in parallel pathways for small regu-latory RNA biogenesis. Dicer-2 (Dcr-2) processeslong-double-stranded precursors and generates siRNAs(19,20) while Dicer-1 (Dcr-1) processes pre-miRNAs intomature miRNAs (20). Importantly, these two enzymesutilize different co-factors for their respective functions.Dcr-1 is associated with the dsRNA-binding proteinLoquacious (Loqs) (21,22) whereas Dcr-2 is associatedwith the dsRNA-binding protein R2D2 (19,23). Incontrast, mammals possess only a single species of Dicer

in association with the dsRNA-binding protein TRBP,which participates in both si- and miRNA biogenesis(24,25). Given this fundamental difference betweenhuman and fly RNAi pathways, we sought to characterizethe mechanism by which highly functional siRNAs areselected in humans.

*To whom correspondence should be addressed. Tel: +1 626 301 8360; Fax: +1 626 301 8271; Email: [email protected]

15101525 Nucleic Acids Research, 2011, Vol. 39, No. 4 Published online 23 October 2010doi:10.1093/nar/gkq846

The Author(s) 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/

by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


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In humans, the HIV-1 TAR RNA-binding protein(TRBP) has been characterized as one of the factors thatassemble Dicer-generated small RNAs into RISC by inter-acting directly with Dicer and Ago2 (2629). TRBP hasbeen shown to have versatile roles in small regulatoryRNA biogenesis. TRBP has high-sequence similarity toDrosophila Loqs and mammalian PACT (30), and is

required for both si- and miRNA silencing (27,29). TheDicer-TRBP interaction is RNA independent (29). TRBPis required for the recruitment of Ago2 to the siRNADicer complex (27) and is proposed to participate incoupling of the RNAi initiation and effector steps.Other studies have shown that MOV10 (31,32) andRNA Helicase A (33) are additional factors that associatewith human RISC, forming an active complex. However,little is known about their modes of action in RNAi.

The selection of the guide strand which serves as asequence-specific RNAi trigger largely determines theefficacy of RNAi (3). Effective siRNAs most often havea thermodynamically unstable 50 guide strand end (34),and often have asymmetric loading of the guide versus

passenger strands (35). In Drosophila, the orientation ofthe guide strand of the siRNA is determined by the Dcr-2/R2D2 heterodimer siRNA binding, with Dcr-2 binding atthe 50-end of the guide strand and R2D2 binding the50-end of the passenger strand (36). R2D2 is also sensitiveto the 50-end stability and the presence of 50 phosphates onthe siRNA (36).

In comparison to observations with the siRNAs, whichoften show a wide range of potency (3739), Dicer pro-cessing of its substrates during the initiation step of RNAiwas shown to result in better programming of RISC(9,19,28). Furthermore the polarity of Dicer processingpreferentially defines which strand serves as the guide(40). Conversely, it is known that Dicer processing is dis-

pensable for RISC assembly and siRNA-mediatedcleavage of target-RNA transcripts when siRNAs wereexogenously introduced into cells lacking functionalDicer (4143). Interestingly, in Drosphila, an organismwhich expresses both siRNAs and miRNAs as regulatorysmall RNAs, the Dcr-2/R2D2 heterodimer was shown toact as a gatekeeper promoting the incorporation ofsiRNAs for assembly with Ago2 while disfavoringmiRNAs as loading substrates for Ago2 (44). Thesorting process depends upon the structure of the smallRNA duplex and not upon Dcr-2 cleavage.

Aside from the appropriate selection of the guide strandfor incorporation into RISC, there is the concern ofoff-targeting in which either the sense or antisense

strand binds to non-targeted sequences triggering inhib-ition of expression of these transcripts (4548). Thereforein order to enhance the efficacy of RNAi, it is essential tobetter understand the mechanism of siRNA selection andstrand incorporation in the RNA-silencing pathway.

Here we show that siRNAs associate with ahigh-molecular weight complex in HEK293 and HCT116whole cell extracts. The degree of this association variesfrom siRNA to siRNA, in some cases dramaticallybetween two siRNAs differing by only a single basepair. We observe that Dicer is the core siRNA-duplex-binding component of the whole cell extract

complex. Dicer preferentially recognizes the 2-nt 30 over-hangs and thermodynamically unstable ends. Since Dicercan bind to a siRNA duplex in either orientation, eitherstrand of the duplex can be selected as the guide strandbased on the thermodynamic end properties of the duplex.Thus, when siRNAs are delivered exogenously to cells,human Dicer (most likely in the form of a complex with

TRBP and Ago2) serves as the primary sensor for theselection of highly functional siRNAs leading to handoffto Ago2. This selective process is based upon the presenceof ribose 2-nt 30 overhangs and the thermodynamic endproperties of the siRNAs. When siRNAs cannot interactwith Dicer, such as in Dicer null cells, they can bypass theDicer-binding step and directly enter RISC. Neverthelesswhen Dicer is present, it serves as a primary sensor forsiRNA selection when the siRNAs contain an appropriatestructure.

MATERIALS AND METHODS

Synthetic siRNAs

All siRNAs used in this study were synthesized by IDT(Coralville, Iowa). The siRNAs were purified usinghigh-performance liquid chromatography. The sequencesare listed in Figures 1 and 3.

Cell extracts

HEK293 cells were grown to confluency in 10-cm dishes,harvested and washed with 1x PBS. The cell pellet wasre-suspended in 0.5 ml of buffer D (20 mM HEPES, pH.7.9, 0.2 mM EDTA, 0.5 mM DTT, 50mM KCl, 10%Glycerol, 0.2 mM PMSF) and sonicated three times for20 s on ice with a Sonifier 450 (Branson, Danbury, CT)

at a setting of 60. The supernatants were collected after15 min of microcentrifugation for direct use in subsequentexperiments.

Dual luciferase reporter assays

To generate reporter plasmids psi-hnRNPH-S (sensereporter) and psi-hnRNPH-AS (antisense reporter), a343-bp PCR fragment of hnRNPH cDNA (Acc.:NM_005520) was cloned in the 30-UTR of the humanizedRenilla luciferase gene in the psiCHECKTM-2 vector(Promega) in either the sense or antisense orientation(40). HCT116 cells at 6080% confluence in 24-wellplates were transfected in duplicate or triplicate with100 ng of reporter DNA, 25200 pM siRNA and 0.5ml

Lipofectamine2000 per well, in a total transfectionvolume of 500ml. Cells transfected with an irrelevantsiRNA were used as mock controls, and an average wascalculated from the replicates to set Renilla/Fireflyluciferase expression to 100%.

For EGFP repression, psi-EGFPS1-S (sense reporter)or psi-EGFPS1-AS was generated by cloning a 245-bpPCR fragment of EGFP in either the sense or antisenseorientation in the 30-UTR of the psiCHECKTM-2 vector(Promega) (40). Forty nanograms of reporter DNA, 20 or200 pM siRNA and 0.5ml Lipofectaimine2000 per wellwere used to transfect HEK293 cells in duplicate at

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6080% confluency in 48-well plates, in a total transfec-tion volume of 200ml. HEK293 cell lysates were collected24-h post-transfection and subjected to Dual Luciferaseassays (Promega). Transfection was done in duplicateand repeated at least three times. Renilla luciferase expres-sion was normalized to internal control Firefly luciferaseexpression. Cells transfected with an irrelevant siRNA

were used as mock controls, and an average wascalculated from the replicates to set Renilla/Fireflyluciferase expression to 100%.

For siDicer, siAgo2 or siTRBP treatment, HEK293cells were seeded on 10-cm dishes and transfectedwith 40-nM siRNA-targeting Dicer, Ago2 or TRBP. Thesequences of the siRNAs used in this study are asfollows: Dicer, 50-UUUGUUGCGAGGCUGAUUCdTdT-30; Ago2, 50-GCACGGAAGUCCAUCUGAAdTdT-30; TRBP, 50-UCUACGAAAUUCAGUAGGAdTdT-30. Forty-eight hours later, the siRNA treatedcells were seeded on either 48-well plates or 6-well platesfor Dual Luciferase assays or RT-qPCR, respectively. Inthe second-round transfection, the same procedure

described above was used for Dual Luciferase assays.Either with or without 20-nM siDicer, siAgo2 orsiTRBP in the second round of transfection producedthe same results.

IC50 measurements

To generate 50% inhibitory concentration (IC50) curves,HEK293 or HCT116 cells at 6080% confluency in48-well plates were transfected in duplicate with 40 ng ofreporter DNA (either psiEGFPS1-S or AS with HEK293cells and psi-H1-S or AS with HCT116 cells), siRNAsranging from 0.2 pM to 15nM in serial dilutions (aminimal of 10 points measurements) and 0.5 mlLipofectaimine2000 per well in a total transfectionvolume of 200ml. Cell lysates were collected 24-hpost-transfection and subjected to the Dual-LuciferaseReporter Assay System (Promega). For each siRNA,transfection was carried out in duplicate and repeated atleast three times. Normalization of Renilla luciferase ex-pression to internal control Firefly luciferase expressionwas as described above. IC50 curves were generated withGraphPad Prism version 5.01 using the dose responsecurve equation: Y= Bottom+(Top Bottom)/(1+10 [(LogIC50 X)HillSlope)] with variable slope.The 95% confidence intervals of IC50 values for siRNAsin this study are listed in Figures 3 and 4.

Recombinant proteins

N-terminally His6x-tagged TRBP2 was expressed from thepET11a (+) vector (Novagen) in the Escherichia coliBL21DE3 strain. Following 4-h induction with 0.5 mMIPTG, purification of His6x-TRBP2 was performed withNickel-Nitrilotriacetic acid (Ni2+-NTA) (the QIA expres-sion system, Qiagen) under denaturing conditions.Efficiency of elution steps was determined with 12%SDSPAGE/coomassie blue staining. The protein wasfurther purified by dialyzing with a 3500 MWCOdialysis cassette (#66110, PIERCE, Rockford, IL),

concentrated with an Amicon Ultra centrifugal filter30KNMWL and stored in buffer D containing 10 mMTCEP. Purity of His-TRBP was also determined byCoomassie blue staining and western-blot analyses (49)with an a-His tag antibody (ab18184, Abcam Inc.,Cambidge, MA). Recombinant human Dicer waspurchased from Ambion, and its concentration was

determined with A205 measurements. Titration-bindingassays were performed to evaluate optimal concentrationsof the recombinant proteins (data not presented), rangingfrom 0.1mg to 5 mg for Dicer and 4 ng to 300 ng for TRBP,2mg of rDicer enzyme and 80 ng of rTRBP were used insubsequent gel shift assays (Figure 5).

Immunodepletion assays

Immunodepletion analyses were carried out as previouslydescribed (49). To immunodeplete Dicer, TRBP or Ago 2from HEK293 whole cell extracts, 2 mg of pre-clearedextract were incubated with each primary antibodya-Dicer (15mg of sc-30226, Santa Cruz Biotechnology,Inc., Santa Cruz, CA), a-TRBP [25ml of TRBP-JBX,(50)], a-Ago2 (15mg of ab57113, Abcam Inc.,Cambridge, MA). In the case of Dicer/TRBP or Ago2/TRBP double immunodepletion, TRBP precipitationwas performed following Dicer or Ago2 precipitations.Pre-immune serum (2.5ml of sc-2007 or sc-2025, SantaCruz Biotechnology, Inc.) was used as a control.Following 2-h incubation, protein A/G plus agarosebeads (Santa Cruz Biotechnology, Inc.) were added andincubated overnight at 4C with gentle agitation. The fol-lowing day, supernatants were collected for additional in-cubation with the beads for 6 h. After removing the beadsby a brief microcentrifugation, buffer D was added to

bring the cell extracts to a final concentration of 4 mg/ml. Of total protein extracts, 15mg were used in the GelShift assays. Of the cell extracts, 150 mg were also used inwestern-blot analyses to evaluate the efficiency of theimmunodepletions. Equal amounts of total protein wereelectrophoresed in SDSPAGE gels (16 cm 16 cm), andeach protein was detected sequentiqlly. After confirmingthe reproducibility of the procedure, the membrane wascut in three pieces to detect Dicer ($215 kDa), Ago2($100 kDa), TRBP ($40kDa) and b-actin ($37 kDa),and the whole membrane was scanned at once for consist-ency using two different wave lengths. Antibodies a-Dicer(ab14601, Abcam Inc.), a-Ago2 (ab57113, Abcam Inc.),a-TRBP (ab42018, Abcam Inc.) or a-b-Actin (A5441,

Sigma-Aldrich, St. Louis, MO) were used to detect thecorresponding proteins. Additionally, the molecularweights of TRBP and Beta-actin are very close. Thus inorder to avoid stripping the membrane between eachprobing, the secondary antibodiesanti-mouse-800nm(610-132-121, Rockland, Gilbertsville, PA) or anti-rabbit-680nm (A21109, Molecular Probes/Invitrogen)were detected at two different wavelengths on anOdyssey Infrared imaging system (LI-COR biosciences,NE). Scanned results in green, red or yellow werevisualized as black and white. The reproducibility of theimmunodepletions was verified four times.

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Gel-shift assay

For incubations with radio-labeled siRNAs, single-stranded siRNAs were 50-end labeled with T4 polynucleo-tide kinase (New England Biolabs) and g-32P-ATP (MPBiochemicals) for 1 h and purified with a G25 column (GEHealthcare). For siRNA duplexes, independently radio-labeled sense and antisense strands were mixed andannealed at 95C in a heat block for 2 min and allowedto cool slowly at room temperature by removing the heatblock from the heat source until they reached ambienttemperature. SiRNA duplexes that were radiolabeledafter annealing yielded identical results. UnlabeledEGFPS1-A, B, hnRNPH-1 and -3 siRNA duplexeswere used as cold competitors. For siRNAs with onlyone 50-end-labeled strand, the sense or antisense strandwas annealed with the unlabeled complementary strandto generate S*/AS or S/AS* EGFPS1A or unlabeledsense and antisense strands were annealed and used as acold competitor. Of total protein in buffer D, 15.0 mg wereincubated with 1 nM (4 104 c.p.m.) of labeled siRNA

duplexes for 30 min at room temperature. In competitionassays, cold siRNA competitors of concentrations at 2, 5,10, 25, 50 or 100 nM were incubated in addition to 1 nMof labeled EGFPS1A siRNA duplex. The samples weremixed with 4X native gel loading dye and resolved in a4.5% non-denaturing polyacrylamide gel (29:1)(2030 cm glass plates, 1.5 mm spacers) with 1X TBEfor 3.0 h at 200V and 4C. The gel was dried at 80Cfor 1 h for autoradiography. Quantifications of theRNAprotein complexes were performed with densito-metric scanning of the gel (Typhoon scanner). Thepercent complex binding was calculated as percentbound [bound/ free+bound) 100] of siRNAs relative toinput siRNA incubated without the cell extract.

Percentage bound of the HEK293 WCE sample was setto 100%, and relative intensities of complexes formed inimmunodepleted cell extracts were calculated accordingly.

For the gel supershift assays, HEK293 cell extract(15.0mg of total protein) was incubated with 1mg ofantibody specific for Dicer (ab14601), Ago1 (07-599,Upstate, Lake Placid, NY), Ago2 (07-590, Upstate) or1ml TRBP antibody Ab672 (51) for 15 min prior to theincubation with siRNA duplexes. The samples wereresolved in a composite polyacrylamide (3%)agarose(0.5%) gel and run for 7 h at 100V at 4C. The gel wasfixed with a 10% Acetic Acid: 10% methanol mix for20 min and dried at 80C for 1 h for autoradiography.The antibodies used in the supershifts were also used in

western blot analyses (49) to detect the proteins in the cellextract. The assays described above were repeatedmultiple times and representative gel figures are shownin this study.

RT-qPCR

Twenty-four hours after the second round of transfectionwith 20-nM siRNA-targeting Dicer, Ago2 or TRBP, and200-pM siEGFPS1A variants, RNA was extracted withRNA STAT60 (TEL-TEST), treated with TurboDNA-Free Kit (Ambion, Austin, Texas) and reverse-transcribed into complementary DNA (cDNA) using

random hexamer primers and Maloney MurineLeukemia Virus (MMLV) reverse transcriptase(Invitrogen, Carlsbad, California). One RNA sample ofeach preparation was processed without MMLV RT asa negative control in subsequent real time PCR reactions.Quantitative analysis of Dicer, Ago2 and TRBP expres-sions was performed by real time PCR SYBR Green I (Bio

Rad) analysis (C1000 Thermal Cycler, Bio Rad, Hercules,California). Dicer, Ago2 and TRBP expression wasdetected using 50 ng of cDNA, amplified with correspond-ing primer sets Dicer-A (50-CATGGATAGTGGGATGTCAC-30), Dicer-B (50-CTACTTCCACAGTGACTCTG-30); Ago2-A (50-CGCGTCCGAAGGCTGCTCTA-30), Ago2-B (50-TGGCTGTGCCTTGTAAAACGCT-30) and 50TRBP (50-GGGCTGCCTAGTATAGAGC-30), 30TRBP-2 (50-GACCCGGAAGGTGAAATTAG-30). GAPDH expression was detected as internalcontrol using 50 ng of cDNA, with primers GAPDH-A(50-CGCTCTCTGCTCCTCCTGTT-3 0) and GAPDH-B(50-CCATGGTGTCTGAGCGATGT-3 0). PCR condi-tions are as follows: (i) Dicer, Ago2 and GAPDH PCR:

95C for 5 min, followed by 40 cycles of 95C for 30 s,60C for 30 s and 72C for 1 min, and (ii) for TRBP andGAPDH: 95C for 5 min, followed by 40 cycles of 95Cfor 1 min, 60C for 1min and 72C for 1 min.

RESULTS

Differential whole-cell-extract complex formation andtarget knockdown correlate with siRNA end structure

To test the structural requirements for binding of siRNAsto components of whole cell extracts and targetknockdown, various 30-end modified versions of a singleEGFP-targeting siRNA (EGFPS1) were incubated in

whole-cell extracts for evaluation of complex formationand in cell culture for target down-regulation assays.The sequence of the antisense (guide) strand of theEGFPS1A variants was kept constant and only thesense strand was varied to create the differing types ofoverhangs (Figure 1a). The results of the binding assaysrevealed that differences in complex formation are sensi-tive to the 30-end structure. When the siRNA duplexes inFigure 1a were incubated with a HEK293 whole-cellextract, the strongest binding was observed with the 2-nt30 overhang (EGFPS1A or 19+2 in Figure 1a and b) andwas reduced with subsequent alterations in the siRNA endstructure. Binding reached


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effects of various structural features, we chose to use con-centrations of 20 and 200 pM, or a concentration at theEGFPS1A IC50 value (Figure 3) versus one 10-fold abovethis. The variant siRNAs were co-transfected with thepsiEGFPS1-S reporter plasmid in HEK293 cells and the

effects on Renilla luciferase activity were monitored. The19+2 siRNA was most potent, consistent with it being thebest binder in the extracts. The other siRNAs followed ingraded fashion with the +1, blunt, 1 and 2 siRNAsbeing progressively worse in target knockdown. These

Figure 1. The effect of 30 overhang on complex formation and RISC function. (a and b) Four variants of EGFPS1A siRNA differing in the numberof nucleotides at 30-end (a) were evaluated for complex formation competence (b) + 2, + 1,+ 0, 1, 2 indicate numbers of nucleotides at the 3 0-endas overhangs [19+2, 20+1, 21+0 (blunt ends), 20 1 (with 1-nt 50 overhangs) and 19 2 (with 2-nt 5 0 overhangs)]. The bottom strands are theantisense sequences and the upper strands the sense sequences. Without HEK293 whole-cell extract, 19+2 was included as a negative control. Theband shifts were quantified by densitometric scanning of the gel (Typhoon scanner) and percent bound siRNAs were calculated by [bound/

(free+bound) 100] of siRNAs relative to input siRNA without cell-extract incubation. The assays were conducted multiple times with similarresults, and a representative gel is shown here. ( c) Dual Luciferase assays. To determine the efficiency of the 3 0-end modified siRNAs in intracellulartarget knockdown efficiency, silencing by the antisense strand of the end modified EGFPS1A siRNA duplexes was assayed in HEK293 cells byco-transfecting the psi-EGFP-S1 sense reporter and 20- or 200-pM siRNAs. Target-specific Renilla luciferase expression was normalized to thecontrol Firefly luciferase expression for all replicates (determined from multiple co-transfections).



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cellular assays were consistent with their binding activitiesin the cell extracts.

We next tested the specificity of complex formation withsingle- or double-stranded siRNAs to determine if thehigh-molecular weight complex observed is an RNAduplex specific (Figure 2). When either the EGFPS1Alabeled sense (S*) or antisense (AS*) strands were testedindividually in the binding assays, no binding to thehigh-molecular weight complex was observed (Figure 2)whereas a faster migrating complex was observed withboth of the single-strand RNAs. We further tested the

duplex requirement of the complex using EGFPS1A con-taining either one (S*/AS or S/AS*) or both (S*/AS*)50-radiolabeled strands. Both S*/AS and S/AS* wereincorporated into the complex, and cold EGFPS1A (S/AS) efficiently competed with the S*/AS* siRNAprotein complex, confirming that the complex in thisstudy is duplex specific (Figure 2a). These observations

demonstrate that the complex formation in this study ispreferentially with the duplex form of siRNAs. Our ob-servations are also consistent with reported observationsthat the human RLC forms exclusively with the duplexform of siRNAs (2729).

To test the complex formation with several differentcanonical siRNAs harboring 19-base duplexes and 2-ntoverhangs, we created a set of ten siRNAs targetingEGFP (EGFPS1-AJ, Figure 3a) in which each sequenceis shifted by 1 nt towards the 50-end of the target RNA.Several, but not all of the siRNAs incorporated publishedsiRNA features that contribute to the overall efficiency ofRNAi (such as an A at position 3 or 19 and 3050%overall GC content in the passenger strand)

(37,39,5254). Duplex end free energiesfrom 2 to 5 bpon both the 50- and 30-endsof the siRNAs were alsocalculated and are listed in Table 1, panel a. When the10 32P-labeled anti-EGFP siRNA duplexes were incubatedin HEK293 cell extracts and subjected to EMSA analyses,the duplexes formed complexes that migrated similarly.However, while the majority of siRNAs formed strongcomplexes, EGFPS1B, I and J formed weak complexes(Figure 3b). We next examined the relative levels ofsiRNA-directed mRNA knockdown using DualLuciferase assays. IC50 values for both sense (S) and anti-sense (AS) targets of the siRNAs were determined usingdose-dependent target knockdown measurements(Figure 3c and Supplementary Figure S1). When the

EGFPS1 siRNAs were co-transfected with Renillaluciferase target reporter plasmidspsiEGFPS1-S orpsiEGFPS1-AS (40)into HEK293 cells, differentialknockdown efficiencies of the siRNAs were observed(Figure 3c). The general trend observed was that thesiRNAs with the stronger complex formation percentages(Figure 3b) had better overall combined IC50 values(Figure 3c). In contrast, the three siRNAs with thepoorest complex formation percentages (EFGPS1B, Iand J, Figure 4b) had high-combined IC50 values(Figure 3c), and in the case of S1J there was nomeasureable target knockdown for the psi-EGFPS1-AStarget (Figure 3c).

As representatives of good and poor binders in the

HEK293 cell extract assay, EGFPS1A and B were subse-quently incubated with HCT116 cell extracts. Consistentwith the observation from the HEK293 cell extract,EGFPS1A and B formed strong and weak complexes, re-spectively, in the HCT116 cell extract (data not shown),validating that the siRNA-binding differentials are notextract dependent.

To further explore the relationship between complexformation in the whole-cell extracts and RNAi efficacy,we created an additional set of siRNAs targeting ahuman gene encoding the heterogeneous nuclearribonucleoprotein (hnRNP) H and tested these for

Figure 2. Duplex specificity of siRNAs for complex formation.(A) Duplex requirement for complex formation. Sense (S*) or antisense(AS*) strand of EGFPS1A siRNA was incubated separately in the cellextract, and their gel shift patterns were compared with the intactEGFPS1A duplex. The duplex specificity of the complex was furthertested with EGFPS1 A siRNA containing either one (S*/AS or S/AS*)or both (S*/AS*) 50-labeled strands (lanes 36). Cold S/AS was addedas a competitor (lane 4). Both S*/AS and S/AS* EGFPS1A wereincorporated into the complex (arrow).



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complex formation in HCT116 whole cell extracts (Figure4). Seven 21-nt-long siRNAs (hnRNP H17) weredesigned with their sequences shifted 1 nt toward the30-end of the target RNA hnRNP H (Figure 5a) (40)and these were evaluated in the extracts by EMSA. Aswith the first set of siRNAs, duplex end-free energiesfrom 2 to 5 bp on both the 50- a n d 30-endsof thesiRNAs were also calculated and are listed in Table 1,

panel b. When the seven 32P-labeled anti-hnRNPHsiRNA duplexes were incubated in HCT116 cell extractsand subjected to EMSA analyses, the duplexes formedcomplexes that migrated similarly (Figure 4b). However,among the seven anti-hnRNP H siRNAs, which wereshifted by a single base from each other, we observed arange of binding efficiencies: hnRNP H1, H2, H4 and H7were effectively incorporated into the complex, whereas

Figure 3. Ribonucleoprotein complex formation and EGFPS1 siRNA silencing. (a) A set of 10 anti-EGFP siRNAs-targeting EGFP Site I (40).Upper strand is sense; bottom strand is antisense. (b) Gel-shift assays. Anti-EGFPS1-A to -J siRNAs were incubated with HEK293 cell extract andresolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1. The assays were conducted multiple times withsimilar results, and a representative gel is shown here. (c) Determination of EGFPS1 siRNA IC50 values. HEK293 cells were co-transfected witheither psiEGFPS1-Sense or -Antisense reporter plasmid and an siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 pointsmeasurements). Cell lysates collected 24-h post-transfection was subjected to the Dual-Luciferase Reporter Assay System (Promega) (dose-dependent

target knockdown measurement curves are shown in Supplementary Figure S1). The n.a. indicates knockdown data were not obtainable for thepassenger strand of S1J.



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complex. We were unable to immunodeplete Ago2 in thecell extract (Id Ago2, Figure 7), suggesting poor recogni-tion of Ago2 by this antibody, perhaps due to interactionsof Ago2 with other proteins in the extracts. Nevertheless,

residual levels of Ago2 in Id Dicer, Id TRBP and Id Dicer/TRBP cell extracts had no effect on complex formation,consistent with the previous report that Ago2 does notassociate with siRNA duplexes by itself (27). It is ofinterest to note that the combined use of anti-Ago2 andTRBP antibodies resulted in depletion of Ago2 in additionto Dicer and TRBP and this also abolished complex for-mation. This result is most likely a consequence of tightinteractions among TRBP, Dicer and Ago2 (27). Mostimportantly we observed loss of siRNA gel shifts whenthe cell extracts were immunodepleted of Dicer, or theDicer/TRBP heterodimer (Figure 7, lanes 46 and 8 in

the left panel, and lanes 35 and 7 in the right panel). Intotal, these observations support Dicer as thesiRNA-binding factor in the cell extracts.

To further explore the role of Dicer in the complex for-mation we replenished the Dicer immunodepleted extractsusing purified recombinant Dicer and/or TRBP and testedfor EGFPS1A complex formation (Figure 8). WhilerTRBP alone had no effect on complex restoration inboth Id Dicer (lane 5) or Id TRBP cell extracts (lane13), addition of rDicer or rDicer in combination withrTRBP (lanes 6, 7, 12 and 14) resulted in restoration ofthe siRNA/complex formation (Figures 8ac).

Figure 4. Ribonucleoprotein complex formation and hnRNPH siRNA silencing. (a) A set of seven hnRNPH siRNAs targeting the hnRNP H site I(40). Upper strand is sense, lower strand is antisense. (b) Gel-shift assays. Anti-hnRNPH siRNAs were incubated with (+) or without () HCT116cell extract and resolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1. The percent bound of each siRNA isshown under the corresponding gel lane. The assays were conducted multiple times with similar results, and a representative gel is shown here.(c) Determination of hnRNPH1 siRNA IC50 values. HCT116 cells were co-transfected with either psi-H1-Sense or -Antisense reporter plasmid andan siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 points measurements). Cell lysates collected 24-h post-transfection wassubjected to the Dual-Luciferase Reporter Assay System (Promega) (Dose dependent target knockdown measurement curves are shown in

Supplementary Figure S2).

Figure 5. Competition assays. The specificity and requirement forsiRNA duplexes for complex formation were examined using competi-tion assays. Duplex requirements for complex formation were testedwith radio-labeled EGFPS1A siRNA mixed with 2-, 5-, 10-, 25-, 50-or 100-fold molar excess of cold EGFPS1A, -B, hnRNPH1 or H3.Quantifications of the RNAprotein complexes were performed usingdensitometric scanning of the gel (Typhoon scanner).



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Given the differential complex formation of several ofthe siRNAs described above, we next asked if Dicer alonecan discriminate binding of a strong versus weak complexforming siRNA. To do this rDicer was incubated with

either the EGFPS1-A or -B siRNA duplexes. As weobserved in the high-molecular weight complex formation,rDicer alone showed differential binding affinities betweenthese two siRNAs, forming a much stronger interactionwith the EGFPS1A than with the EGFPS1B duplex(Supplementary Figure S3). Although rDicer aggregatedand was poorly resolved in the composite gel, the differ-

ential binding was reproducible.To dissect the relative roles of Dicer, Ago2 and TRBP

in selective incorporation of siRNAs in vivo, we usedRNAi to knockdown each of these proteins and assayedfor EGFPS1A-mediated target knockdown using thepanel of EGFPS1A siRNAs that differ in their 30-endstructures (depicted and tested in Figure 1). If Dicerbinding initially determines the 30-end structure andguide strand selection, the differential efficiencies of theEGFPS1A variants should be absent in Dicer knockdowncells. Of course knockdown of Ago2 would also reduceRNAi overall since it is required for the RNAi effectorstep of cleaving the target transcripts. We compared theknockdowns of EGFP via the EGFPS1A panel inHEK293 in cells pre-treated with siRNAs targetingDicer, Ago2 or TRBP (Figure 9). Consistent with a keyrole for Dicer in siRNA selection, the previously observeddifferences in knockdown efficiencies of the EGFPS1Avariants were substantially reduced in cells pre-treatedwith the anti-Dicer siRNA. In these cells, we determinedthat levels of Dicer and Ago2 expression were repressed$85% and 65%, respectively (Supplementary Figure S4).Although TRBP mRNA levels were reduced by $60%,the effects of this reduction on the differential knockdownefficiencies by the EGFPS1A panel were minimal, which is

Figure 7. Analysis of RISC components required in high-molecular-complex formation. Gel-shift assays. EGFPS1A duplex was incubated withHEK293 cell extracts immunodepleted of Dicer (Id Dicer), TRBP (Id TRBP), Dicer and TRBP (Id Dicer/TRBP), Ago2 (Id Ago2) or Ago2 andTRBP (Id Ago2/TRBP). Evaluation of immunodepletion of RISC proteins with western blot analyses was shown on the right. Of Id cell extracts,150mg were loaded on a 8% SDSPAGE gel. Pre-immune serum was used as an immunodepletion control.

Figure 6. Analysis of proteins comprising the complex. Supershift assays. a-Dicer, a-TRBP, a-Ago2 or a-Ago1 antibodies wereadded to the HEK293 cell extract and incubated for 15 min prior toaddition of either EGFPS1A or hnRNPH1 siRNA. Western analysesof proteins detected with the antibodies were also shown on the right.Of total protein, 50 mg for detection of Dicer and Ago1 or 150mg forAgo2, TRBP and b-actin was loaded on 8% SDSPAGE gels.



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consistent with our EMSA results (Figures 7 and 8). Tofurther validate these results, we also tested the effect ofDicer knockdown on siRNA-triggered gene silencing bycomparing knockdown efficiencies of hnRNPH5 and H6in siDicer-treated HEK293 cells (Supplementary FigureS5). When the siRNAs were co-transfected with thepsi-H1-S or psi-H1-AS reporter plasmid at 200 pM, asym-metric knockdowns of the sense or antisense targets by thesiRNA duplexes were observed; however knockdown

efficiencies of hnRNPH5 and H6 siRNAs were substan-tially reduced in siDicer treated cells (SupplementaryFigure S5). Our observations indicate that the overallefficacies of the siRNAs were diminished in the Dicerknockdown cells, consistent with previous observations(27,56), which is most likely a consequence of disruptionof the RLC.

To discriminate the selective binding aspect of Dicerfrom direct incorporation of the siRNAs into Ago2, wemodified EGFPS1A to generate an asymmetric 27/25-nt-long Dicer substrate form (DsiRNA)a blunt endwith two deoxyribonucleotide bases at the 30-end of the

passenger strandforcing the duplex to be bound andundergo Dicer cleavage activity. EGFPS1B was alsomodified to a DsiRNA form for direct comparison, andthe IC50 values were determined for both DsiRNAs(Figure 10a and b). For both EGFPS1A and BDsiRNAs, the strand harboring the 2-nt 30 overhangwas predominantly selected as the guide strand (IC50,

Dsi-S1A-AS =31.9 pM, IC50, Dsi-S1A-Sense =101 pM; IC50,

Dsi-S1B-AS =27.5 pM, IC50, Dsi-S1B-Sense= 433 pM), con-

sistent with previous observations (40). In contrast, thedominant selection of the antisense strand (relative tothe sense EGFP target) was absent or less pronouncedwith the use of 19+2 siRNAs. Both the dsiRNAs andsiRNAs form dicer-dependent complexes in whole-cellextracts (57) (Supplementary Figure S6). These resultssupport a model in which the orientation of Dicerbinding to its substrates sets the preference for whichstrand will be chosen to act as the guide strand. Takentogether, our observations suggest that the initial DicerDsiRNA interaction is an important determinant for es-tablishing siRNA-strand selection in RISC and ultimately

Figure 8. Analysis of siRNARISC complex formation. (a) Reconstitution of the siRNA complex with recombinant proteins. Id Dicer (left) or IdTRBP (right) cell extracts were mixed with 2 mg of recombinant Dicer, 80 ng of recombinant TRBP or both proteins to rescue complex formation.(b and c) Percent-bound-reconstituted siRNA complexes were calculated relative to the HEK293 WCE which was set as 100%.



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the potency of the siRNAs. The Dicer substrates forceDicer to enter the RNA via its PAZ domain and the

2-base 3

0

overhang of the Dicer substrate whereas 19+2siRNAs allow Dicer to bind from either end using the2-base 30 overhang for PAZ domain interactions.

DISCUSSION

We have used siRNA binding to a high-molecular-weightcomplex in whole-cell extracts to better understand whatcellular factors are involved in the initial binding and dis-criminatory selection of highly functional versus poorlyfunctional siRNAs. In particular, we have been interestedin what happens to siRNAs following transfection intocultured cells. We find that the extent of siRNA complexformation reflects siRNA-directed overall target

knockdown efficiency in cultured cells. Using antibodiesto core RNAi proteins to determine the key proteins ofthe complex, we find that it minimally consists ofDicer/TRBP, presumably as the pre-RLC. Our observa-tions suggest that human RISC silencing efficiency withexogenously supplied siRNAs reflects the extent ofsiRNA-pre-RLC formation.

Previously, mammalian Dicer was shown to be dispens-able for RISC assembly and siRNA-mediated cleavage oftarget RNA transcripts (4143). In contrast, the absenceof a functional mouse Dicer resulted in a lack ofshRNA-mediated RNAi (43), showing that Dicer-

cleavage activity was necessary to generate the siRNAsfrom this precursor. The lack of a Dicer requirement

for pre-formed siRNA function led to the conclusionthat only Ago2 was required for siRNA selection.Conversely, two groups demonstrated that the absenceof Dicer abolished gene silencing effects (27,56). Ourresults suggest that when present, Dicer itself bindssiRNAs that mimic the products of Dicer cleavage(19 bp + 2-nt 30 overhangs) and Dicer can serve as agate-keeper discriminating between potent andnon-potent siRNAs by selective binding of the siRNAsin the pre-RLC.

Schwarz et al. reported that 24 bp on both ends of asiRNA are important for their relative loading efficiencyinto RISC in Drosophila (35). Our calculated duplexend free energies for siRNAs used in this study

(Table 1) are consistent with these observations.Moreover, we observed that in general, siRNAs withthermodynamically unstable base pairs at both ends ef-fectively form high-molecular-weight complexes in cellextracts and have better overall silencing efficiencies.These observations suggest that the thermodynamicproperties of 24bp on both ends of effective siRNAsare used as functional determinants by the pre-RLCand determine overall silencing efficiency in humans aswell.

It is widely known that siRNAs targeting the sameregion of a target mRNA can have a wide range of

Figure 9. The effect of 30 overhang on RISC function in Dicer kd, Ago2 kd and TRBP kd cells. Efficiencies of target downregulation by fourvariants of EGFPS1A siRNA (Figure 1) were evaluated using Dual Luciferase assays with siDicer, siAgo2 or siTRBP treated HEK293 cells. Todetermine effects of Dicer, Ago2 or TRBP knockdowns on the silencing activity of the siEGFPS1A variants described in Figure 1, HEK293 cellswere treated with 40 nM siDicer, siAgo2 or siTRBP for 2 days prior to the co-transfection. For the second round of transfection, Dicer kd, Ago2 kdor TRBP kd cells were transfected with the psi-EGFP-S1 sense reporter and 200pM siEGFPS1A variants. Target-specific Renilla luciferase expres-sion was normalized to the control Firefly luciferase expression for all replicates (determined from four co-transfections).



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potencies. To circumvent this obstacle, reported siRNAfeatures such as an A at position 3 or 19 and 3050%overall GC content in the passenger strand (37,39,5254)are widely used to predict potent siRNAs in addition to

thermodynamic end properties. However, the importanceof the AU base pair at these positions remained elusive.Using two sets of siRNAs targeting EGFP site I andhnRNP H site I (40), we observed that siRNAs containingan A:U base pair at positions 1 and/or 19 result in moreeffective binding to the high-molecular-weight complexthan those with C:G pairs at these positions. Thisfinding is consistent with the previous report of Katohand Suzuki (58) which suggested that an AU base pairat these positions makes the siRNA a good substrate forthe pre-RLC interaction and selection as an effectivesiRNA for target downregulation.

The manipulations of the 2-nt 30 overhangs, whichresulted in reductions in target downregulation(Figure 1) verify that the presence of a 1- or 2-nt 30

overhang is required not only for efficient binding to the

high-molecular-weight complex, but is also requiredfor efficient target downregulation. Previously, Pellinoet al. (55) reported that the complex observed withHEK293 cell extracts contained Dicer. Our siRNA gelshift and binding assays along with the immunodepletionassays in HCT116 and HEK293 cell extracts demonstratethat Dicer is required for siRNA binding in these extracts,a result which is consistent with reports that the initialsiRNADicer interaction is via the 2-nt 30 overhang(59,60).

The marked differences in Dicer/TRBP binding tosiRNAs and the correlations with efficacy of target

Figure 10. Comparative analyses of guide strand selection for 21-mer and 25/27-mer Dicer substrate siRNAs. Target knockdowns for sense andantisense strands of siRNA EGFPS1A (a) and EGFPS1B (b) in 21-mer and Dicer substrate formats were carried out using co-transfections of thesiRNAs with either the psi-EGFP-S sense reporter (red) or psi-EGFP-AS antisense reporter (black) in HEK293 cells. For both targets the 21-ntEGFPS1 siRNAs (left) or 25/27 DsiRNAs (right) were tested at various concentrations. Target-specific Renilla luciferase expression was normalizedto the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). Sequences and IC50 values of each siRNAare shown. For the Dicer substrate siRNAs, the lower case letters represent deoxyribonucleotide containing bases which block Dicer entry.



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knockdown in cell culture, even for siRNAs differing byonly a single nucleotide in sequence, point to the import-ance of understanding how siRNAs are selected for entryinto RISC. Understanding the rules for effective siRNAdesign is further complicated by the fact that siRNAswhich do not bind to Dicer can also be incorporatedinto RISC (4143). We propose though that when Dicer

is present it will preferentially bind and select siRNAsharboring structurally favorable features, and this inter-action leads to the subsequent handoff step to Ago2.

Selective binding of Dicer to siRNAs could potentiallydetermine the strand to be incorporated as a guide strand.The binding of the PAZ domain to a Dicer substrateallows the catalytic domain to interact with the oppositeend of the dsRNA. Studies of the interaction of the humanDicer catalytic site with the PIWI domain of Ago2 (61)and the binding of the Archaeoglobus fulgidus PIWIprotein with the 50-end of the guide strand in the crystalstructure (62) suggest the coupling of the initiation andeffector steps of RNAi. The handoff of the guide strandfrom Dicer to Ago2 via the Dicer-catalytic domain couldexplain not only why Dicer processing of longer precur-sors often increases the efficacy of processed siRNAs(40,63), but also why the 50-end of the siRNA withinRISC leads to and determines the stable association ofRISC and the target RNA (64). In this regard, Dicer sub-strates in the studies of Rose et al. (40) have a blunt endwith two deoxybases at the 30-end of the passenger strand,which precludes Dicer PAZ domain interactions from thatend of the duplex. The polarity of Dicer entry from theend harboring the 2-base 30 overhang results in substan-tially reduced function of the passenger strand relative tothat observed with the corresponding 19+2 siRNA(Figure 10). Moreover, a single-nucleotide shift in the

siRNA relative to the target site also resulted in a strongbias in guide strand selection for the dsiRNAs asdemonstrated by EGFPS1A-25/27 (IC50,

Dsi-S1A-Sense= 101pM) and EGFPS1B-25/27 DsiRNAs(IC50, Dsi-S1B-Sese = 433 pM) (Figure 10). Thus, Dicerinteraction with the 2-nt 30 overhang is an importantstep in Dicer binding, and for 19+2 siRNAs Dicer canbind in two orientations. Nevertheless, the thermodynam-ic end properties of the siRNAs also influence Dicerbinding as we have demonstrated.

Recent single-particle EM analyses of theRLC-containing Dicer/TRBP/Ago2, show that Ago2 isassociated with the C-terminal domain of Dicer, whileTRBP is associated with the N-terminal or helicase

domain of Dicer (65,66). This structural configurationwould facilitate Dicer handoff of the 50-end of the guidestrand to Ago2. Our observations that Dicer binds select-ively to 2-base 30 overhangs, most likely via the PAZdomain, would orient the enzyme such that theC-terminal portion of the enzyme is associated with the50-end of the guide strand to facilitate handoff of the guidestrand to Ago2. An important observation is that the sta-bility of Dicer binding to an siRNA is not solely mediatedby the 30 2-base overhang, but obviously involves inter-actions with the duplex itself. This is made evident by thefact that many of the siRNAs used in our binding analyses

contain the 2-base overhang but are not bound strongly tothe Dicer-containing complex.

Thus far, the binding efficiency of Dicer/TRBP topre-miRNAs appears to be maintained regardless ofwhether or not the pre-miRNA can be cleaved by Dicer(67). Our work and that of Pellino et al. (55) demonstratethat Dicer/TRBP can bind to siRNAs as well. However,

these observations are in contrast to a previous study thathuman Dicer is incapable of siRNA binding (68).One possible explanation for this discrepancy is that thesiRNA used in the previous study may fall into the groupof siRNAs that are weak binders to the pre-RLCsimilarto the poor binding siRNAs tested in our study.

While immunodepletion of Dicer (Id Dicer) left-residualTRBP, Id of TRBP resulted in nearly complete depletionof Dicer in the HEK 293 cell extracts, as did co-Id of Dicer/TRBP. These results indicate that the majority of Dicer isbound to TRBP and that distinctive roles of Dicer andTRBP augment each other to function as the pre-RLC.Id of Ago2/TRBP resulted in immunodepletion of Dicer,TRBP and Ago2 (Figure 7), suggesting that TRBP func-

tions as a bridge between Dicer and Ago2.We have demonstrated that for many siRNAs what we

deemed as the passenger strand could be more effective attarget knockdown than the desired antisense guide strand.Since Dicer/TRBP binding to a siRNA duplex determinesthe strand to be selected as the guide strand, off-targetingwill often be a consequence of both strands beingincorporated into RISC. Thus the two strands of asiRNA can also compete with one another for incorpor-ation into RISC following Dicer selection, thereby poten-tially reducing the potencies of both.

In conclusion our results demonstrate that human Dicerplays an important role in selection of efficacious siRNAsvia direct binding to siRNAs that harbor certain structuralfeatures, which include the 2-nt 30 overhangs and thethermodynamically unstable base pairing at the ends ofthe duplex. These results are similar yet distinct fromDrosophila in which R2D2 and Dcr-2 bind to the thermo-dynamically stable and unstable ends of siRNA duplexes,respectively. Our results demonstrate that the human Dicerenzyme can recognize and selectively bind siRNAs withthermodynamically favorable termini. Our results demon-strate that an often overlooked feature of 19+2 siRNAs isthe ability of both strands to be chosen for RISC entry.This is sometimes observed for miRNAs as well, but oftenonly one strand serves as the guide. In this regard, Dicersubstrate siRNAs mimic the miRNA pathway, providingstrong polarity for guide strand selection, perhaps a usefulattribute for minimizing off target effects and competitionbetween the two strands for entry into RISC.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

The authors acknowledge members of the Rossi labora-tory for helpful discussions and support, especially Lisa



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Scherer for the critical reading of this article. They aregrateful for the suggestions of Y. Chen, R.J. Lin,J Shively and J. Termini.

FUNDING

Norwegian Research Council (a postdoctoral fellowshipto M.A.); Arnold and Mabel Beckman Foundation (toD.K.); National Institutes of Health (grants AI29329,AI42552 and HL074704 to J.J.R.). Funding for openaccess charges: National Institutes of Health NationalHeart Lung and Blood Institute and National Instituteof Allergy and Infectious Diseases.

Conflict of interest statement. M.A.B. is employed by IDTDNA, which manufactures siRNAs and dicer substratesiRNAs. J.J.R. is chairman of the SAB at DicernaPharmacueticals which uses Dicer substrate siRNAs.

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